The present invention is directed to a method of producing multilamellar coalescence vesicles (MLCVs) which contain a high incorporation of biologically active compounds, using small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) without steps involving multiple freeze-thawing cycles, using organic solvents or dehydration of the vesicles.
The present invention also is directed to the MLCVs produced by the present method. These MLCVs possess advantageous properties of containing higher amounts of surface and total biologically active compounds, without the use of human serum albumin (HSA), as compared to prior art multilamellar vesicles (MLVs).
Liposomes are known to be useful as carriers of biologically active compounds which facilitate the delivery of these compounds to the body. Liposomes have been evaluated as potential drug delivery systems to introduce biologically active compounds into cells. See Poznansky and Juliano, Pharmacol. Rev. 36, 277-336 (1984); B. E. Ryman et al., Essays in Biochemistry, 16, 49 (1980). Several routes of administration have been used for the administration of liposomes, for example, intravenous, subcutaneous, intraperitoneal, and oral delivery. See Gregoriadis and Allison, eds., Liposomes in Biological Systems, John Wiley and Sons, New York (1980) at pages 153-178. An important advantage of liposomal delivery is the change in tissue distribution and binding properties as compared to the free forms of the bioactive ingredient, resulting in an enhanced therapeutic index and decreased toxicity. For example, decreased nephrotoxicity has been associated with the use of liposomes containing amphotericin B or cyclosporin A. See G. Lopez-Berestein, Ann. Int. Med., 105, 130 (1985) and Hsieh et al., Transplantation Proceedings, Vol. XVII, 1397-1400 (1985). Also, reduced cardiotoxicity and nephrotoxicity are associated with liposomes containing doxorubicin and cisplatin, respectively, as compared to the free forms of the drugs. See Rahman et al., Cancer Res., 42, 1817 (1982); and Forssen et al., Cancer Res., 43, 546 (1983).
It is known that, under appropriate conditions, phospholipid dispersions can spontaneously reform, in the presence of water, into closed membrane systems. Electron microscopy reveals that these closed structures are made of a number of concentric bilayers or lamellae composed of phospholipid molecules, and are known as liposomes. The usefulness of liposomes as a model membrane system arises from the fact that, as the dry phospholipids undergo a defined sequence of molecular rearrangements, there is an opportunity for an unrestricted entry of hydrophilic solutes into the interlamellae space. Similarly, sequestration of hydrophobic solutes occurs within the hydrophobic bilayers. The result is a delivery system that can contain varying amounts of cytokines or other biologically active compounds, depending on the type of interaction between the solute and the phospholipid assembly.
Many methods have been proposed for the preparation of liposomes. The classical method of making prior art liposomes, MLVs containing a biologically active compound, is to mix a lipid in an organic solvent, remove the solvent from the solution, leaving a residue, suspend the residue in a buffer containing a biologically active compound, agitate and homogenize the suspension until the MLVs which contain the biologically active compound are formed, and isolate the resulting MLVs. See Bangham et al. (1974) In Methods in Membrane Biology (Korn, E., ed.), pp 1-68, Plenum Press, N.Y.
One of the most widely used techniques is known as the thin film method, which involves the aqueous hydration of a dried lipid film. See Bangham et al. Briefly, lipids of the desired composition, in solution with an organic solvent, are dried in the form of a thin film on the walls of a round-bottomed flask. A biologically active compound can be included in the film at this stage. The dry film is hydrated by adding a suitable aqueous phase and gently swirling the flask. With a hydrophilic biologically active compound, an aqueous solution containing the biologically active compound is used for hydration. MLVs are formed by this procedure.
Although MLVs are produced and used in medical applications, a major problem in the manufacture of MLVs is the use of organic solvents to dissolve the lipids. Further, many biologically active compounds are incompatible with organic solvents, and the removal of organic solvents from these preparations is difficult and tedious. Additionally, to form MLVs with high entrapment of biologically active compound, the solution must be subjected to repeated freeze-thawing cycles. Large scale freeze-thawing is difficult to carry out, especially under sterile conditions. Further, to produce MLVs under sterile conditions, it is necessary to sterilize the lipid prior to placing it into solution. This sterilization process may result in the breakdown of the lipids, resulting in the formation of by-products.
The process of the present invention provides a method of producing MLCVs, which has none of the limitations of the prior art methods, and several advantages over the prior art methods. The process of the present invention allows the improved entrapment of solutes. These solutes can be biologically active compounds or any compound, such as HSA, mannitol, or glycerol, which can be entrapped by the liposomes, MLCVs, of the present invention. Examples of additional biologically active compounds useful in the present invention are pharmaceutical peptides, proteins, antigens and drugs or any biologically active compound that can be incorporated into a liposome for delivery to a subject.
The present process results in the production of MLCVs without the use of organic solvents while supplying the means for sterilizing the lipid in an aseptic process by filter sterilization. The process of the present invention can be used easily to produce a small scale production run as well as a large production run while maintaining a simple manufacturing scheme. Moreover, the MLCVs of the present invention are unique structurally in that they possess a varying degree of partially coalesced vesicles in addition to numerous lamellae. Further, the present method produces MLCVs that possess a consistent size and consistent distribution of biologically active compound with less variability than obtained using the prior art methods.
MLCVs made by the method of the present invention contain a greater amount of biologically active compound as a result of enhanced entrapment. The present MLCVs possess a greater amount of surface biologically active compound and exhibit a greater recovery of biologically active compound than the prior art MLVs. Therefore, the present method results in enhanced recovery and incorporation of biologically active compounds as compared to the prior art. In regard to the present invention, the term recovery is defined as the percent of output over input; that is, the amount of the biologically active compound that is present in products and unreacted starting materials with the remainder being lost in processing. The term incorporation as used in the present invention is defined as the percent of output that is entrapped in liposomes and is no longer free.
The process of the present invention is referred to as a coalescence process because the produced MLCVs are produced as a result of rupturing and resealing of bilayers accompanied by leakage of internal contents. This process differs from vesicle fusion in which the combining of vesicles is accompanied by the mixing of internal contents with no or minimal leakage. See Gingell, D. and Ginsberg, L. (1978), In: Membrane Fusion (Poste, G. and Nicolson, G. L., eds.), pp.791-833, Elsevier/North-Holland Biomedical Press, NY.; Szoka, F. (1987), In: Cell Fusion (Sowers, A. E., ed.), pp. 209-240, Plenum Press, NY; Nir, S., Wilschut, J. and Bentz, J. (1982), Biochim. Biophys. Acta 688:275-278; Poste, G and Nicolson, G. L. (1978) Membrane Fusion, Elsevier/North-Holland Biomedical Press, NY.
Further, the MLCVs of the present invention have sizes (average diameters) of about 1000 to 5000 nm. However, the average size of the MLCVs of the present invention may be as small as at least about 100 nm or greater; e.g., at least 200 nm or greater. Prior art methods which use fusion of SUVs below the phase transition temperature result in unilamellar vesicles having average diameters of less than 100 nm. Liposomes of an average diameter greater than 100 nm have a greater accumulation in the lung, liver, spleen and lymph nodes than smaller liposomes. These liposomes with an average diameter greater than 100 nm are often used to target organs for prophylactic and therapeutic treatment. See Jackson (1981), Drug Med. Disp. 9: 535-540.
The method of the present invention is directed to producing multilamellar coalescence vesicles (MLCVs) containing a biologically active compound by hydrating at least one powdered lipid in an aqueous buffer at a temperature above the phase transition temperature of the highest melting lipid to form multilamellar vesicles (MLVs), reducing the size of the MLVs to about 20-400 nm to produce small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) or a mixture thereof, and incubating the SUVs, LUVs or mixture thereof with at least one biologically active compound in an aqueous solution under sufficient conditions to form MLCVs containing said at least one biologically active compound. The method of the present invention is performed without the use of an organic solvent, a freeze-thawing step or a dehydration step. The size reducing step of the method comprises exposing the MLVs to a high shear force which is effected by one or more of sonication, homogenization or extrusion. The method further includes sterile filtering the SUVs, LUVs or mixture thereof prior to mixing the SUVs, LUVs or mixture thereof with the biologically active compound(s).
The method of the present invention discloses the production of MLCVs containing increased amounts of biologically active compounds. These MLCVs have an average diameter of at least 100 nm or greater, preferably in the range of 1000 to 5000 nm, and are produced without the use of steps involving freeze-thawing, organic solvents or dehydration.
The method of the present invention includes the steps of hydrating powdered lipid(s) with an appropriate buffer in a temperature jacketed mixing vessel at a temperature above the phase transition temperature of the highest melting lipid. Then, the size of the vesicles is reduced from the micron range to about a 20-400 nm range. These vesicles are small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUV) or a mixture thereof, and are produced by known standard methods that utilize high shearing forces, such as sonication, homogenization or extrusion. Homogenizers particularly useful in the present invention are high pressure homogenizers, such as those manufactured by Gaulin Rannie or Microfluidics. This latter step is also carried out at a temperature above the phase transition temperature of the lipid mixture. The resulting SUVs and/or LUVs are then sterile filtered into a mixing vessel which is also maintained at a temperature above the phase transition temperature of the highest melting lipid. The biologically active compound(s) and any necessary excipients are added through a sterilizing filter and mixed while dropping the temperature to the phase transition temperature of the highest melting lipid, or maintaining the temperature, preferably between the pretransition and main transition temperature range. The incubation may also be performed below the pretransition, below the subtransition or above the main transition temperature of the lipid system. Then, the mixture is incubated for a period of time from minutes up to days. During this time the SUVs and/or LUVs are coalesced to form larger vesicles, MLCVs, which are generally have about 100 nm or greater average diameter.